Genome Projects and Gene technologies Flashcards
what are the three methods for producing fragments of DNA ?
-using reverse transcriptase
-using restriction endonuclease enzymes
-using a gene machine
what is recombinant DNA?
DNA of two different species/two types of organisms that have been combined
How can you make DNA Fragments with reverse transcriptase?
-mRNA that was transcribed for the desired gene used
-mRNA acts as a template for production of single stranded stranded cDNA using reverse transcriptase
-cDNA is isolated by hydrolysis of the mRNA with DNA helicase
-double stranded DNA is formed on the template of cDNA using DNA polymerase
How can you make DNA fragments with restriction endonuclease enzymes and what are they ?
-restriction endonucleases are enzymes that can cut DNA at a specific base sequence
-the base sequence they recognise is called the recognition or restriction site
-the enzyme cuts DNA between two specific bases (cleavage site)
-this produces sticky ends
-which result in one strand of the DNA fragment being longer than the other strand
- make it easier to insert the desired gene into another organism’s DNA as they can easily form hydrogen bonds with the complementary base sequences on other pieces of DNA
sticky ends are used in genetic engineering. Explain how
-joining two pieces of DNA
-by complementary base pairing
what are endonucleases?
enzymes produced by bacteria to cut up viral DNA as a defensive measure
Explain what sticky ends are and their importance in recombinant DNA technology.
-DNA is cleaved at the restriction site
-restriction endonucleases cuts out the DNA fragment
-sticky ends are staggered pieces of DNA that are complementary to each other
-complementary bases of the two sticky ends pair up
-DNA ligase joins sugar phosphate backbone
-allows us to bind DNA from one organism to another provided
-the same restriction endonucleases is used
How do we use a gene machine to produce DNA fragments?
-sequence of bases determined
-triplets worked out and fed into computer
-computer designs oligonucleotides which are assembled into the desired gene
-PCR replicates gene
-gene into plasmid using sticky ends
-gene check using sequencing techniques
positive evaluation of using the gene machine
-quick
-no introns=prokaryotes cant splice
-any sequence of nucleotides
-accurate
why are the same restriction endonucleases used to cut the DNA and the vector?
ensures the sticky ends of the gene are complementary to each other
what are the two ways of gene cloning ?
-in vivo- inside living organisms
-in vitro- outside living organisms
recall how a gene is inserted into a plasmid vector (in vivo 1.)
-sticky ends are complementary since same RE ends are used
-promoter and terminator regions added
-DNA ligase forms two phosphodiester bonds
State the steps of introducing DNA to host cells (in vivo 2.)
-host bacterial cells are placed into cold calcium chloride solution to make cell walls more permeable
-plasmids are added and mixture is heat shocked wich encourages the cells to take in the plasmid
State the steps in identifying transformed cells (in vivo 3.)
-replica plating (making copies of colonies on agar plate)
-grow in tetracycline
-marker gene can code for antibiotic resistance so only transformed cells that have the marker gene will survive and grow
-or marker genes can code for fluorescence so when the agar plate is placed under a UV light only transformed cells with fluoresce
What are the three stages of PCR
-denaturation
-annealing
-extension
